G. Bauw

Characterization of a rice gene showing organ-specific expression in response to salt stress and drought.

Protein changes induced by salinity stress were investigated in the roots of the salt-sensitive rice cultivar Taichung native 1. We found eight proteins to be induced and obtained partial sequences of one with a molecular mass of 15 kilodaltons and an isoelectric point of 5.5. Using an oligonucleotide probe based on this information, a cDNA clone, salT, was selected and found to contain an open reading frame coding for a protein of 145 amino acid residues. salT mRNA accumulates very rapidly in sheaths and roots from mature plants and seedlings upon treatment with Murashige and Skoog salts (1%), air drying, abscisic acid (20 microM), polyethylene glycol (5%), sodium chloride (1%), and potassium chloride (1%). Generally, no induction was seen in the leaf lamina even when the stress should affect all parts of the plant uniformly. The organ-specific response of salT is correlatable with the pattern of Na+ accumulation during salt stress.

An S18 ribosomal protein gene copy at the Arabidopsis PFL locus affects plant development by its specific expression in meristems.

In Arabidopsis, mutation at PFL causes pointed first leaves, reduced fresh weight and growth retardation. We have cloned the wild‐type PFL gene by T‐DNA tagging, and demonstrate that it complements the mutant phenotype. PFL codes for ribosomal protein S18, based on the high homology with rat S18 and on purification of S18‐equivalent peptides from plant ribosomes. pfl represents the first mutation in eukaryotic S18 proteins or their S13 prokaryotic counterparts, involved in translation initiation. Arabidopsis contains three S18 gene copies dispersed in the genetic map; they are all transcribed and code for completely identical proteins. No transcript is detected from the mutated gene, S18A. The activity of the S18A promoter is restricted to meristems, with a markedly high expression at the embryonic heart stage, and to wounding sites. This means that plants activate an extra copy of this ribosomal protein gene in tissues with cell division activity. We postulate that in meristematic tissues plants use transcriptional control to synthesize extra ribosomes to increase translational efficiency. In analogy with this, an additional, developmentally regulated gene copy might be expected for all ribosomal proteins.

Protein-Electroblotting and Microsequencing in Establishing Integrated Human Protein Databases

Proteins, which are characteristic for a specific state of differentiation, the transformed phenotype or pathological conditions of human cells and tissues were identified by computer analyzed two-dimensional gel electrophoresis. Sequenceable amounts of protein were collected from multiple gels with a gel-concentration device, enabling the elution and concentration of more than twenty protein spots, suspended in 1 ml of sample buffer. The eluted protein was concentrated in a new gel in a very small spot and then electroblotted onto polybase-coated glass-fiber or polyvinylidene-difluoride membranes and in situ digested. The released peptides were separated by micro-bore or narrow-bore reversed phase HPLC and immediately collected on polyethylenimine-coated glass-fiber discs for sequencing. These variations of previously developed methods allowed us to work at higher sensitivity. The procedure is currently being used to try out a systematic analysis of human proteins recovered from two-dimensional gels.

A thiol protease and an anionic peroxidase are induced by lowering cytokinins during callus growth in Petunia

We previously identified a group of proteins that increase early in Petunia hybrida calli subcultured on a low-cytokinin medium, unlike the calli subcultured on a high-cytokinin medium. The calli on the low-cytokinin medium do not regenerate (J.-P. Renaudin, C. Tournaire, B. Teyssendier de la Serve [1991] Physiol Plant 82: 48–56). Two of these proteins, P21 and P17, have been identified by peptide sequencing and cloned. P21 is highly homologous to a group of thiol proteases, including barley aleurain, rice oryzain [gamma], Arabidopsis SAG2, and mammalian cathepsin H. P17 is highly homologous to a group of anionic peroxidases from potato and tomato. A study of their expression in two P. hybrida lines, PC6 and St40, which differ in their ability to regenerate, showed that the genes for P21 and P17 are differentially expressed depending on the type and the age of the organ, with the highest expression in senescing leaves and in aged calli. The data are in favor of these genes being associated with an early step of senescence, which may be due, in part, to a reduction in total cytokinin. The two Petunia lines are, thus, functionally different concerning the action of cytokinin in two developmental phenomena: in vitro organogenesis and senescence.

Protein Blotting from Polyacrylamide Gels on Glass Microfiber Sheets: Acid Hydrolysis and Gas-Phase Sequencing of Glass-Fiber Immobilized Proteins

The transfer onto immobilizing membranes of proteins which are separated on polyacrylamide gels (popularly referred to as the Western blot) has become an important tool in protein chemistry (for a comprehensive review see [1]). Originally, transferred proteins were covalently bound onto the membrane [2, 3], but this method showed low coupling yields and was therefore replaced by the introduction of microporous nitrocellulose membranes to which proteins could be bound by noncovalent interactions [4]. This principle was later improved by using a nylon-based, positively charged membrane [5]. Although proteins immobilized in this way could be easily assayed for their antigenicity or their enzymatic or lectin-binding activity [1], the membranes used for these analyses were sensitive to the chemical treatments necessary for acid hydrolysis or for the Edman-de-gradation-based chemistry. In order to combine the simple, fast, inexpensive, high-resolution purification procedure of polyacrylamide gel electrophoresis with the recently developed technique of protein gas-phase sequencing [6], it was necessary to introduce a support which is both able to bind every kind of protein eluting from a gel, and resistant against chemicals and solvents used in Edman chemistry.

The role of vacuolar and secreted pathogenesis-related ß (1–3)-glucanases and chitinases in the defence response of plants

SummaryUpon infection of a plant by a pathogen, a series of drastic metabolic changes occur within the plant. One characteristic feature of this defence response is the synthesis of the so-called pathogenesis-related (PR) proteins. We have studied the nature, structure, and subcellular localization of the different PR proteins upon salicylic acid treatment and Pseudomonas syringae infection of Nicotiana tabacum plants. In both test systems, we could demonstrate that the PR protein fraction of tobacco consists of at least 20 to 25 different proteins, including s (1–3)-glucanases, chitinases, peroxidases, thaumatin-like proteins, the PR1 class proteins and a proteinase inhibitor-like protein. Moreover, several classes of these PR proteins segregate into specific vacuolar and secreted isoforms. Here, we present a model which could explain the role of the compartimentalized PR s (1–3)-glucanases and chitinases within the regulation of the defence response.